Oral delivery of macromolecules

ABSTRACT

Polysaccharides, which are widely used as an anticoagulation drugs, especially heparin, are clinically administered only by intravenous or subcutaneous injection because of their strong hydrophilicity and high negative charge. Amphiphilic heparin derivatives were synthesized by conjugation to bile acids, sterols, and alkanoic acids, respectively. These heparin derivatives were slightly hydrophobic, exhibited good solubility in water, and have high anticoagulation activity. These slightly hydrophobic heparin derivatives are efficiently absorbed in the gastrointestinal tract and can be used in oral dosage forms. Methods of using these amphiphilic heparin derivatives and similarly modified macromolecules for oral administration are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of co-pending U.S. Ser. No.09/845,827, filed Apr. 30, 2001, which is a continuation-in-part of U.S.Ser. No. 09/300,173, filed Apr. 27, 1999, now U.S. Pat. No. 6,245,753,which applications are hereby incorporated by reference herein in theirentireties, including but not limited to those portions thatspecifically appear hereinafter, the incorporation by reference beingmade with the following exception: In the event that any portion of theabove-referenced applications are inconsistent with this application,this application supercedes said above-referenced applications.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] This invention relates to derivatives of macromolecules,including polysaccharide derivatives, having increased hydrophobicity ascompared to the unmodified macromolecules or polysaccharides. Moreparticularly, the invention relates to oral delivery and absorption ofhydrophobized macromolecules and amphiphilic polysaccharide derivatives,such as amphiphilic heparin derivatives, wherein the bioactivity of themacromolecule or polysaccharide is preserved. In preferred embodimentsof the invention, the hydrophobized macromolecules and amphiphilicpolysaccharide derivatives have a molecular weight of greater than 1000,yet are absorbed after oral administration.

[0004] Heparin is a polysaccharide composed of sulfated D-glucosamineand D-glucuronic acid residues. Due to its numerous ionizable sulfategroups, heparin possesses a strong electronegative charge. It is also arelatively strong acid that readily forms water-soluble salts, e.g.heparin sodium. It is found in mast cells and can be extracted from manybody organs, particularly those with abundant mast cells. The liver andlungs are especially rich in heparin. The circulating blood contains noheparin except after profound disruption of mast cells. Heparin has manyphysiological roles, such as blood anticoagulation, inhibition of smoothmuscle cell proliferation, and others. In particular, heparin is apotent anticoagulant agent that interacts strongly with antithrombin III(ATIII) to prevent the formation of fibrin clots. Heparin is one of themost potent anticoagulants used for treatment and prevention of deepvein thrombosis and pulmonary embolism. In vivo, however, applicationsof heparin are very limited. Because of its hydrophilicity and highnegative charge, heparin is not absorbed efficiently from the GI tract,nasal or buccal mucosal layers, and the like. Therefore, the only routesof administration used clinically are intravenous and subcutaneousinjections. Moreover, since heparin is soluble in relatively fewsolvents, it is hard to use for coating surfaces of medical devices orin delivery systems.

[0005] To improve the properties of heparin, R. J. Linhardt et al., 83J. Pharm. Sci. 1034-1039 (1994), coupled lauryl (C₁₂) and stearyl (C₁₈)groups to single heparin chains, resulting in a derivatized heparinhaving increased hydrophobicity but with low anticoagulation activity.This result demonstrated that coupling a small linear aliphatic chain toheparin was ineffective in enhancing the hydrophobicity of heparin whilepreserving activity. Thus, known heparin derivatives have beenineffective in preserving anticoagulation activity.

[0006] T. M. Rivera et al., Oral Delivery of Heparin in Combination withSodium N-[8-(2-Hydroxybenzolyl)amino]caprylate: PharmacologicalConsiderations, 14 Pharm. Res. 1830-1834 (1997), disclosed thepossibility of oral delivery of heparin using heparin mixed with sodiumN-[8-(2-hydroxybenzolyl)amino]caprylate. M. Dryjski et al.,Investigations on Plasma Activity of Low Molecular Weight Heparin afterIntravenous and Oral Administrations, 28 Br. J. Clin. Pharma. 188-192(1989), described the possibility of oral absorption of low molecularweight heparin using enhancers.

[0007] It is generally recognized that molecules having a molecularweight greater than 1000 are poorly absorbed in the gastrointestinal(GI) tract after oral administration. For example, J. G. Russell-Jones,Carrier-mediated Transport, Oral Drug Delivery, in 1 Encyclopedia ofControlled Drug Delivery 173, 175 (Edith Mathiowitz ed. 1999), statedthat the work of W. Kramer et al., 269 J. Biol. Chem. 10621-10627(1994), suggested that the maximal size of a peptide that could betransported via the bile acid transporter was four amino acids, or about600 Da. As another example, P. W. Swaan et al., Enhanced TransepithelialTransport of Peptides by Conjugation to Cholic Acid, 8 BioconjugateChemistry 520-525 (1997), reported that bile acid conjugates with up to6 amino acids (i.e., about 900 Da) showed affinity for the intestinalbile acid transporter, but the only 6-amino-acid bile acid conjugatetested was not transported by the bile acid carrier.

[0008] In view of the foregoing, it will be appreciated that developmentof a method for obtaining absorption of macromolecules having amolecular weight greater than 1000 after oral administration would be asignificant advancement in the art. It will also be appreciated thatdevelopment of a method for obtaining absorption of hydrophobized oramphiphilic heparin derivatives after oral administration would beanother significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION

[0009] It is an object of the present invention to provide a method forobtaining absorption of molecules having a molecular weight greater than1000 after oral administration.

[0010] It is also an object of the invention to provide a-method forobtaining blood anticoagulation by oral administration of amphiphilicheparin derivatives.

[0011] It is still another object of the invention to provide heparinderivatives that can be absorbed from the GI tract, thereby facilitatingoral delivery for preventing blood coagulation.

[0012] It is yet another object of the invention to provide heparinderivatives comprising heparin coupled with a bile acid, such asdeoxycholic acid or glycocholic acid, or a hydrophobic agent, such ascholesterol, or an alkanoic acid.

[0013] These and other objects can be addressed by providing a method oftreating a patient in need of anticoagulation therapy comprising orallyadministering an effective amount of a composition comprising heparincovalently bonded to a hydrophobic agent selected from the groupconsisting of bile acids, sterols, and alkanoic acids, and mixturesthereof. The composition can also include a pharmaceutically acceptablecarrier.

[0014] In one preferred embodiment of the invention the hydrophobicagent is a bile acid selected from the group consisting of cholic acid,deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholicacid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholicacid, glycocholic acid, taurocholic acid, glycodeoxycholic acid,glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid,hyodeoxycholic acid, and mixtures thereof, and the like.

[0015] In another preferred embodiment of the invention, the hydrophobicagent is a sterol selected from the group consisting of cholestanol,coprostanol, cholesterol, epicholesterol, ergosterol, ergocalciferol,and mixtures thereof, and the like.

[0016] In still another preferred embodiment of the invention, thehydrophobic agent is an alkanoic acid comprising about 4 to 20 carbonatoms. Preferred alkanoic acids include butyric acid, valeric acid,caproic acid, caprylic acid, capric acid, lauric acid, myristic acid,palmitic acid, stearic acid, and mixtures thereof, and the like.

[0017] Preferably, the heparin comprises a molecular weight of at leastabout 3000, and more preferably at least about 6000. In certainpreferred embodiments, the heparin comprises a molecular weight lessthan about 12,000.

[0018] Another preferred embodiment of the invention comprises a methodfor enhancing oral administration of a macromolecular agent comprising:

[0019] (a) conjugating the macromolecular agent to a hydrophobic agentselected from the group consisting of bile acids, sterols, alkanoicacids, and mixtures thereof, and the like to result in a hydrophobizedmacromolecular agent; and

[0020] (b) orally administering an effective amount of the hydrophobizedmacromolecular agent to a patient in need thereof.

[0021] Preferably, the macromolecular agent is a member selected fromthe group consisting of heparin, heparan sulfate, sulfonylpolysaccharide, heparinoids, polysaccharide derivatives, and mixturesthereof, and the like. In another preferred embodiment of the invention,the macromolecular agent is a peptide, such as insulin or calcitonin.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0022]FIG. 1 shows clotting time profiles as measured by aPTT assay ofheparin-DOCA conjugate after oral administration in rats: □-100 mg/kgraw heparin (control), ⋄-physical mixture of heparin (200 mg/kg) andDOCA (200 mg/kg); ▾-50 mg/kg heparin-DOCA conjugate, ▴-80 mg/kgheparin-DOCA conjugate, □-100 mg/kg heparin-DOCA conjugate, and ◯-200mg/kg heparin-DOCA conjugate; data are plotted as mean±SD, n=9.

[0023]FIG. 2 shows concentration profiles as measured by FXa assay ofheparin-DOCA conjugate after oral administration in rats: □-100 mg/kgraw heparin (control), ▾-50 mg/kg heparin-DOCA conjugate, ▴-80 mg/kgheparin-DOCA conjugate, □-100 mg/kg heparin-DOCA conjugate, and ∘-200mg/kg heparin-DOCA conjugate; data are plotted as mean±SD, n=9.

[0024]FIG. 3 shows clotting time profiles in rats of heparin-DOCAconjugates as a function of the mole ratio of DOCA to heparin: ▾-rawheparin, ▴-2.5 mole ratio, □-5.0 mole ratio, and 0-10.0 mole ratio; dataare plotted as mean±SD, n=9.

[0025]FIG. 4 shows clotting time profiles in rats of heparin derivativesas a function of the hydrophobic agent conjugate to heparin:▾-heparin-lauric acid conjugate, ▴-heparin-palmitic acid conjugate,□-heparin-cholesterol conjugate, and ∘-heparin-DOCA conjugate; data areplotted as mean±SD, n=9.

[0026]FIG. 5 shows micrographs of hematoxylin and eosin stainedgastrointestinal tissues that were isolated from rats after oraladministration of 100 mg/kg of heparin-DOCA conjugate: panels A, B, C.and D show cross sections of the stomach after 0, 1, 2, and 3 hours,respectively; panels E, F, G, and H show cross sections of the duodenumafter 0, 1, 2, and 3 hours, respectively; panels I, J, K, and L showcross sections of the jejunum after 0, 1, 2, and 3 hours, respectively;and panels M, N, 0, and P show cross sections of the ileum after 0, 1,2, and 3 hours, respectively; the original magnification was 100× in allpanels.

[0027]FIG. 6 shows electron micrographs of membrane or microvilli ingastrointestinal tissues isolated from rats after oral administration of100 mg/kg of heparin-DOCA conjugate: panels A, B, C. and D show crosssections of the stomach after 0, 1, 2, and 3 hours, respectively; panelsE, F, G, and H show cross sections of the duodenum after 0, 1, 2, and 3hours, respectively; panels I, J, K, and L show cross sections of thejejunum after 0, 1, 2, and 3 hours, respectively; and panels M, N, 0,and P show cross sections of the ileum after 0, 1, 2, and 3 hours,respectively; the original magnification was 25,000×in all panels.

[0028]FIGS. 7A and 7B show clotting time profiles (FIG. 7A) andconcentration profiles (FIG. 7B) of heparin-DOCA conjugates after oraladministration in rats: ▴-LMWH(3K)-DOCA; ∘-LMWH(6K)-DOCA; □-heparin-DOCA(also referred to herein as UFH-DOCA).

[0029]FIGS. 8A and 8B show clotting time profiles (FIG. 8A) andconcentration profiles (FIG. 8B) of LMWH(6K)-DOCA after oraladministration in rats: ▴-20 mg/kg of LMWH(6K) control; □-100 mg/kg ofLMWH(6K) control; ▴-20 mg/kg LMWH(6K)-DOCA; □-50 mg/kg LMWH(6K)-DOCA;◯-100 mg/kg LMWH(6K)-DOCA.

DETAILED DESCRIPTION

[0030] Before the present methods for obtaining absorption of orallydelivered hydrophobized macromolecules and amphiphilic polysaccharidecompositions are disclosed and described, it is to be understood thatthis invention is not limited to the particular configurations, processsteps, and materials disclosed herein as such configurations, processsteps, and materials may vary somewhat. It is also to be understood thatthe terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

[0031] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a bile acid” includes a mixture of two or more ofsuch bile acids, reference to “an alkanoic acid” includes reference toone or more of such alkanoic acids, and reference to “a sterol” includesreference to a mixture of two or more sterols.

[0032] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0033] As used herein, “bile acids” means natural and syntheticderivatives of the steroid, cholanic acid, including, withoutlimitation, cholic acid, deoxycholic acid, chenodeoxycholic acid,lithocholic acid, ursocholic acid, ursodeoxycholic acid,isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid,dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and mixturesthereof, and the like.

[0034] As used herein, “sterols” means alcohols structurally related tothe steroids including, without limitation, cholestanol, coprostanol,cholesterol, epicholesterol, ergosterol, ergocalciferol, and mixturesthereof, and the like.

[0035] As used herein, “alkanoic acids” means saturated fatty acids ofabout 4 to 20 carbon atoms. Illustrative alkanoic acids include, withoutlimitation, butyric acid, valeric acid, caproic acid, caprylic acid,capric acid, lauric acid, myristic acid, palmitic acid, stearic acid,and mixtures thereof, and the like.

[0036] As used herein, “hydrophobic heparin derivative” and “amphiphilicheparin derivative” are used interchangeably. Heparin is a veryhydrophilic material. Increasing the hydrophobicity of heparin bybonding a hydrophobic agent thereto results in what is termed herein anamphiphilic heparin derivative or hydrophobic heparin derivative. Eitherterm is proper because the heparin derivative has increasedhydrophobicity as compared to native heparin and the heparin derivativehas a hydrophilic portion and a hydrophobic portion and is, thus,amphiphilic.

[0037] As used herein, “aPTT” means activated partial thromboplastintime, and “FXa” means factor Xa.

[0038] As used herein, “DOCA” means deoxycholic acid, and “heparin-DOCA”means a conjugate of heparin and deoxycholic acid.

[0039] As used herein, “macromolecule” means polypeptide,polysaccharide, and nucleic acid polymers with a molecular weighttypically greater than 1000.

[0040] As used herein, “peptide” means peptides of any length andincludes proteins. The terms “polypeptide” and “oligopeptide” are usedherein without any particular intended size limitation, unless aparticular size is otherwise stated. Typical of peptides that can beutilized are those selected from group consisting of oxytocin,vasopressin, adrenocorticotrophic hormone, epidermal growth factor,prolactin, luliberin or luteinising hormone releasing hormone, growthhormone, growth hormone releasing factor, insulin, somatostatin,glucagon, interferon, gastrin, tetragastrin, pentagastrin, urogastroine,secretin, calcitonin, enkephalins, endorphins, angiotensins, renin,bradykinin, bacitracins, polymixins, colistins, tyrocidin, gramicidines,and synthetic analogues, modifications and pharmacologically activefragments thereof, monoclonal antibodies and soluble vaccines. The onlylimitation to the peptide or protein drug which may be utilized is oneof functionality.

[0041] As used herein, “effective amount” means an amount of apharmacologically active agent that is nontoxic but sufficient toprovide the desired local or systemic effect and performance at areasonable benefit/risk ratio attending any medical treatment. Thus, forexample, an effective amount of a heparin-DOCA conjugate is an amountsufficient to provide a selected level of anticoagulation activity.

[0042] It is well known that heparin is used as an antithrombogenicagent to prevent blood coagulation. Heparin is highly hydrophilicbecause of a high density of negative charges such as are provided bysulfonic and carboxylic groups. Due to this hydrophilicity, heparin isusually administered by intravenous or subcutaneous injection. Heparinderivatives with slightly hydrophobic properties or amphiphilicproperties and with high bioactivity are described herein. Hydrophobicagents, such as bile acids, e.g. deoxycholic acid (DOCA); sterols, e.g.cholesterol; and alkanoic acids, e.g. lauric acid and palmitic acid,were coupled to heparin. Both deoxycholic acid and cholesterol arenon-toxic since they are naturally occurring compounds found in thebody. The amine groups of heparin were coupled with carboxyl groups ofthe hydrophobic agents. The end carboxylic groups in DOCA, lauric acid,and palmitic acid were used directly for the coupling reaction, whilethe hydroxy group of cholesterol was activated by reaction withchloroacetic acid before coupling. It was determined that conjugatingsuch hydrophobic moieties to the amine groups of heparin had little orno effect on heparin bioactivity. The coupling between heparin andhydrophobic agents was confirmed by detecting the resulting amide bondby FT-IR and ¹³C-NMR analysis.

[0043] The yield of the coupling reaction was about 70 to 80% and wasnot significantly changed by changing the hydrophobic agents or feedmolar ratios. In the case of the heparin-DOCA conjugate, as the feedratio was increased, the amount of DOCA in the conjugate was alsoincreased. The weight % of DOCA in heparin-DOCA was 24% when the feedmolar ratio of heparin to DOCA was 1:200. This molar ratio was very highcompared to the ratio of amine groups in heparin to DOCA. Therefore,this feed ratio is estimated as an excess amount of DOCA.

[0044] The hydrophobic heparin derivatives according to the presentinvention would have many medical applications. For example, thehydrophobic heparin can be administered orally. The oral administrationof heparin can greatly extend the usage of heparin as an oralanti-coagulant drug. The heparin derivative is formulated with apharmaceutically acceptable carrier such as is well known in the art. Byway of further example, hydrophobic heparin derivatives can be used as acoating material for medical devices such as catheters, cardiopulmonarybypass circuits, heart lung oxygenators, kidney dialyzers, stent orballoon coating for preventing restenosis, and the like. The hydrophobicheparin derivative is typically mixed with a carrier, and then coated onthe surface of the medical device by a film casting technique such as iswell known in the art.

[0045] After modification, heparin-hydrophobic agents were also found tohave a tendency in fast protein liquid chromatography (FPLC®) to exhibithydrophobic interactions with hydrophobic media, as shown bychromatography on Phenyl Sepharose® (eluting in ammonium sulfate bufferrather than phosphate buffer). These heparin derivatives showed enhancedbinding affinity when compared to unmodified heparin. The increasedinteraction of modified heparin derivatives with Phenyl Sepharose® isattributable to its enhanced hydrophobicity, the result of thehydrophobic functional groups present. These results suggest hydrophobicheparin can be obtained by conjugating a bile acid, sterol, or alkanoicacid to heparin. In solubility tests, polar solvents or organic solventswere suitable to dissolve the heparin-hydrophobic agent conjugates. Forexample, the heparin-deoxycholic acid conjugate showed good solubilityin 65% acetone solution (35% water). Finally, it was determined thatbioactivity of modified heparin derivatives was not appreciablyinfluenced by conjugation with hydrophobic agents. The role of ahydrophobic agent conjugated to heparin was studied with respect to twobiological activities of heparin as determined by anticoagulation andfactor Xa assays. Although hydrophobicity is associated with a somewhatreduced anticoagulant activity and antifactor Xa activity, the decreaseof bioactivity was not considered serious. These results indicate thatblocking the amine groups of heparin had little effect on itsbioactivity. The bioactivity of heparin in heparin-hydrophobic agentconjugates exhibited a progressive reduction, however, when the amountof hydrophobic agent in the conjugate exceeded 20 wt. %. At less than 20wt. % of hydrophobic agent in the conjugates, the bioactivity of theconjugates was greater than 80% of the bioactivity of unmodifiedheparin. It is suggested that 80% of bioactivity in hydrophobic heparinis enough to support bioactivity in medical applications.

EXAMPLE 1

[0046] Synthesis of Heparin-DOCA Conjugates. Five ml ofN-hydroxylsuccinimide (HOSu, 92 mg/5 ml) in dimethylformamide (DMF) wasmixed with 5 ml of dicyclohexylcarbodiimide (DCC) (165 mg/5 ml) in DMF,followed by adding 5 ml of DOCA (196 mg/5 ml) in DMF. The mole ratio ofDOCA, HOSu, and DCC was 1:1.6:1.6. The concentrations of HOSu and DCCwere slightly higher than that of DOCA to activate DOCA completely. Theresulting solution was reacted for 5 hours at room temperature undervacuum, and then the byproduct dicyclohexylurea (DCU), whichprecipitated during the reaction, was removed. The unreacted DCC wasremoved by adding a drop of distilled water and filtering. The remainingHOSu was also removed by adding 15 ml of distilled water. The activatedDOCA was precipitated and then lyophilized. The activated DOCA was thendissolved in DMF and reacted with heparin for 4 hours at roomtemperature. The amounts of heparin used in such reactions ranged from40 to 400 mg. After reaction, there were two types of products: a watersoluble product and a water-insoluble product. These products wereseparated by filtration through a 0.45 μm membrane filter, and thewater-insoluble product (i.e., activated DOCA) was dried in a vacuumoven. The water-soluble product (i.e., heparin-DOCA) was dialyzed for 1day against water using a membrane (MWCO 3,500), and then heparin-DOCAwas freeze dried.

[0047] The synthesized heparin-DOCA was further purified by reversephase chromatography. A phenyl-Sepharose CL-4B column (HR 16/30 I.D.)was washed with 100 ml of distilled water, 400 ml of 50 mM phosphatebuffer (pH 7.0), 40 ml of 50 mM phosphate buffer (pH 7.0) containing 1.7M ammonium sulfate, and 40 ml of 50 mM phosphate buffer, respectively.Five milliliters of the heparin-DOCA solution (1 mg/ml) was loaded inthe column and the heparin-DOCA was fractionated by step elution with anammonium sulfate solution. Elution was carried out with phosphate bufferfor 20 minutes, followed by the ammonium sulfate solution (50 mMphosphate buffer (pH 7.0)+1.7 M ammonium sulfate) with the flow rate of1 ml/min. Heparin-DOCA was eluted in the ammonium sulfate solution. Thepurified heparin-DOCA solution was dialyzed in distilled water andlyophilized.

[0048] The heparin derivatives prepared according to this procedure werecharacterized by FT-IR and NMR according to methods well known in theart to prove the successful coupling between heparin and the hydrophobicagent. Y. Lee, H. T. Moon & Y. Byun, Preparation of Slightly HydrophobicHeparin Derivatives Which Can Be Used for Solvent Casting in PolymericFormulation, 92 Thromb. Res. 149-156 (1998).

EXAMPLE 2

[0049] Preparation of Heparin-Cholesterol Conjugates. The hydroxyl groupof cholesterol was activated by reaction with chloroacetic acid toresult in a free carboxyl group. This modified cholesterol was thenreacted with HOSu and DCC in 10 ml of DMF according to the procedure ofExample 1. The mole ratio of cholesterol, HOSu, and DCC was 1:1.6:1.6and reaction was for 5 hours at room temperature. To remove theunreacted DCC and HOSu, water was added and the solution was filteredwith a 0.45 μm membrane. Next, the activated cholesterol was reactedwith heparin solution for 4 hours. Two products, a water-soluble productand a water-insoluble product, were obtained from the reaction. Theseproducts were treated according to the procedure described above inExample 1.

EXAMPLE 3

[0050] Synthesis of Heparin-Alkanoic Acid Conjugates. Lauric acid andpalmitic acid were coupled to heparin according to the procedure ofExample 1. The carboxyl group of the alkanoic acids were coupled withamine groups of heparin to form amide bonds. Coupling agents were alsoHOSu and DCC.

EXAMPLE 4

[0051] Synthesis of Heparin-Cholic Acid Conjugates. In this example, theprocedure of Example 1 is followed except that cholic acid issubstituted for DOCA.

EXAMPLE 5

[0052] Synthesis of Heparin-Chenodeoxycholic Acid Conjugates. In thisexample, the procedure of Example 1 is followed except thatchenodeoxycholic acid is substituted for DOCA.

EXAMPLE 6

[0053] Synthesis of Heparin-Ergosterol Conjugates. In this example, theprocedure of Example 2 is followed except that ergosterol is substitutedfor cholesterol.

EXAMPLE 7

[0054] Synthesis of Insulin-DOCA Conjugates. In this example, theprocedure of Example 1 is followed except that insulin is substitutedfor heparin. The amine groups of insulin, i.e., GlyAl, PheB1, andLysB29, are coupled with carboxyl groups of DOCA.

EXAMPLE 8

[0055] Synthesis of Calcitonin-DOCA Conjugates. In this example, theprocedure of Example 1 is followed except that calcitonin is substitutedfor heparin. An amine group of calcitonin is coupled to a carboxyl groupof DOCA.

EXAMPLE 9

[0056] Measurement of Bioactivity of Heparin Derivatives. Forheparin-DOCA, heparin-cholesterol, and heparin-alkanoic acid preparedaccording to the procedures of Examples 1-3, the production yield,molecular weight, and binding mole ratios between heparin andhydrophobic agents varied according to the mole ratio of reactants. Theyield of heparin-DOCA conjugates was in the range of 71 to 77%. Theamount of hydrophobic agent in modified heparin derivatives wascalculated by subtracting the molecular weight of heparin (i.e., 12,386daltons as determined by light scattering) from the measured molecularweight of each heparin derivative. As the feed mole ratio of deoxycholicacid to heparin was increased from 1:6 to 1:200, the amount of DOCA inheparin-DOCA conjugates was increased from 7 to 24%. For theheparin-cholesterol conjugates, the yield also was in the range from 73to 78%. The amount of cholesterol in such hydrophobic heparinconjugates, however, was slightly lower than the amount of DOCA inheparin-DOCA conjugates. In heparin-lauric acid and heparin-palmiticacid conjugates, similar amounts of alkanoic acid were coupled toheparin.

[0057] Anticoagulant activities of heparin derivatives were determinedby aPTT assay and FXa chromogenic assay. The activities of heparinderivatives in the prevention of fibrin clot formation were measured byaPTT assay. Each of the platelet-poor-plasma containing heparinstandards (0.1 to 0.7 U/ml, 0.1 ml) and plasma samples containingheparin derivatives (0.1 ml) was incubated with 0.1 ml of APTT reagentfor 2 min at 37° C. After the incubation, 0.1 ml of 0.02 M calciumchloride was added, and the time was recorded from this point until thefibrin clot was formed. The bioactivity of the heparin derivative wascalculated by comparing the clotting time with the heparin standardcurve. The clotting time was linearly proportional to the activity ofheparin in the plasma.

[0058] The activity and the concentration of heparin derivatives werealso determined by FXa chromogenic assay. Each of the heparin standardsand plasma samples containing heparin derivatives (25 μl) was mixed with200 μl of AT III solution (0.1 IU/ml), where the ATIII concentration wasin excess of the heparin concentration. This solution was incubated at37 for 2 min, and 200 μl of FXa (4 nkcat/ml) was added. The resultingsolution was then incubated for an additional 1 min. The concentrationof FXa was also in excess of the heparin concentration. FXa substrate(200 μl, 0.8 μmol/ml) was then added and incubated at 37 for 5 min. Thereaction was terminated by adding 200 μl of acetic acid (50% solution).The bioactivity and the concentration of heparin in the plasma samplewere calculated from the absorbance at 405 nm. These data are summarizedin Table 1. TABLE 1 Compound Mole ratio^(a) Bioactivity (IU/mg) Mol. Wt.Heparin — 140 12,386 Heparin-DOCA 2.5 130 ± 1.0 13,357 Heparin-DOCA 5113 ± 2.8 14,403 Heparin-DOCA 10 100 ± 4.3 16,320 Heparin-cholesterol4.5 122 ± 6.7 13,791 Heparin-lauric acid 5 118 ± 5.0 13,400Heparin-palmitic acid 4.4 123 ± 2.7 13,500

EXAMPLE 10

[0059] Oral Administration of Heparin-DOCA. Sprague-Dawley rats (male,250-260 g) were fasted for 12 hours before dosing. The rats wereanesthetized with diethyl ether and then were administered a single doseof heparin derivative through an oral gavage that was carefully passeddown the esophagus into the stomach. The gavage was made of stainlesssteel with a blunt end to avoid causing lesions on the tissue surface.The solution containing the heparin derivative was prepared in a sodiumbicarbonate buffer (pH 7.4). The total administered volume ofheparin-derivative-containing solution was 0.3 ml. The dose amount wasvaried at 50, 80, 100, and 200 mg/kg, respectively. There were 9 rats ineach group. Blood (450 μl) was collected serially by capillary from theretro-orbital plexus at each time point and directly mixed with 50 μl ofsodium citrate (3.8% solution). The blood samples were immediatelycentrifuged at 2500×g and 4° C. for 5 minutes. The clotting time and theconcentration of heparin derivative in the plasma were measured by aPTTassay and FXa assay, respectively.

[0060] The absorption of heparin-DOCA in the GI tract was determinedaccording to the dose amount in the range of 50 to 200 mg/kg. In thisexperiment, the mole ratio of coupled DOCA to heparin in theheparin-DOCA conjugate was 10. When raw heparin was administered orallyto rats, the clotting time, measured by aPTT assay, was about 18 secondsand this value did not change over time. The average value of thebaseline was 18 seconds, indicating that the raw heparin was notabsorbed in the GI tract. When the physical mixture or admixture ofheparin and DOCA was administered orally, the aPTT value was about 20seconds, and this value did not change over time. On the other hand,when heparin-DOCA conjugate was orally administered, the clotting timeincreased as shown in FIG. 1. Since the blood sampling was carried outat one-hour intervals and the maximum clotting time was shown at theone-hour time point, the real maximum clotting time could not bedetermined. However, the clotting time at one hour was linearlyincreased with the increase of dosage. When heparin-DOCA conjugate wasgiven at 50, 80, 100, and 200 mg/kg, the clotting times at one hour were25.8±2.6, 43.1+4.0, 51.2+9.3, and 136±33 seconds, respectively. Whenheparin-DOCA conjugate was administered at 200 mg/kg, the clotting timeat one hour increased greatly, above 7-times the baseline. Since thetherapeutic window of heparin is 1.5 to 2.5 times the baseline, thetherapeutic effect can be seen at an 80-100 mg/kg dose. Therefore, theheparin-DOCA conjugate greatly enhanced the absorption of heparin in theGI tract, in contrast to DOCA mixed with heparin in a physical mixture,which did not enhance heparin absorption.

[0061] The concentration of heparin-DOCA conjugate in the plasma wasdetermined by FXa assay, as shown in FIG. 2. The concentration profilesof heparin-DOCA conjugate over time were similar to the results of theaPTT assay shown in FIG. 1. The concentration of absorbed heparin-DOCAincreased with the increase of the dosage. The therapeutic target rangewas 0.1 to 0.2 IU/ml. For a 200 mg/kg does of heparin-DOCA conjugate,the mean concentration peak at one hour was about 9-10 times thebaseline and the concentration at that time was about 1.0 IU/ml. Theplasma concentration of heparin-DOCA conjugate returned to the baselineafter 3 hours. Therefore, the absorption of heparin-DOCA in the GI tractwas confirmed.

EXAMPLE 11

[0062] Heparin-DOCA Conjugate Absorption in the GI Tract of Rats. Todetermine the absorption of heparin-DOCA conjugate in the GI tract as afunction of the ratio of DOCA to heparin, heparin-DOCA conjugates weresynthesized with DOCA:heparin mole ratios of 2.5, 5.0, and 10.0, asdescribed in Example 1. As shown in Table 1, the bioactivity ofheparin-DOCA conjugates decreased slightly as the mole ratio of DOCA toheparin increased. However, since the molecular weight of heparin-DOCAincreased as the mole ratio of DOCA to heparin increased, thebioactivity of heparin-DOCA conjugates as a function of mole ratiodecreased only about 5%. That is, the bioactivities of heparin andheparin-DOCA conjugate (10:1 mole ratio) were 1,734 and 1,632±7 IU/mol,respectively.

[0063]FIG. 3 shows the change in the clotting time according to thecoupled mole ratio of DOCA to heparin. In this experiment, the dosage ofheparin-DOCA conjugate was 100 mg/kg. When the mole ratio of the coupledDOCA to heparin increased, the bioactivity of heparin-DOCA conjugateslightly decreased, as shown in Table 1, whereas the maximum clottingtime increased. This result indicates that the heparin-DOCA conjugatefacilitated absorption of heparin in the GI tract of rats.

EXAMPLE 12

[0064] Effect of Hydrophobic Agent Coupled to Heparin on GI Absorptionin Rats. To show the effect of a hydrophobic agent coupled to heparin onGI absorption, heparin-DOCA, heparin-cholesterol, heparin-palmitic acid,and heparin-lauric acid prepared according to Examples 1-3 were tested.As shown in Table 1, the mole ratio of hydrophobic agent to heparin wascontrolled in the range of 4 to 4.5. The bioactivities of these heparinderivatives were similar to each other, i.e., in the rage of 113 to 123IU/mg. FIG. 4 shows the absorption values obtained after oraladministration of 100 mg/kg of these heparin derivatives. Maximumclotting times at one hour after administration, as measured by the aPTTassay, were 32+6.1 seconds for heparin-cholesterol, 29+8.3 seconds forheparin-palmitic acid, and 25.9+6.6 seconds for heparin-lauric acid. Thecarbon numbers of cholesterol, palmitic acid, and lauric acid were 24,16, and 12, respectively, and the hydrophobicity of the hydrophobicagent is proportion to the number of carbon atoms. Thus, the maximumclotting time increase with the hydrophobicity of the coupledhydrophobic agent. This result indicated that the hydrophobicity of theheparin derivative was an important property for increasing theabsorption of heparin in the GI tract. Even though cholesterol is morehydrophobic than DOCA, however, heparin-DOCA conjugate exhibited ahigher clotting time than heparin-cholesterol conjugate. Possibleexplanations for this observation include (1) the amphiphilic propertiesof heparin-DOCA conjugate, which may improve the permeability of theheparin derivative in the GI wall, and (2) the interaction between theDOCA moiety of the heparin-DOCA conjugate and the DOCA receptors in theGI wall, especially in the ileum, which might increase the adhesion ofheparin-DOCA conjugate to the GI wall, thereby increasing theprobability of absorption.

EXAMPLE 13

[0065] Histological Evaluation of GI Tract. In this example heparin-DOCAconjugate was administered to rats by oral gavage according to theprocedure of Example 10. The mole ratio of coupled DOCA to heparin inthe heparin-DOCA conjugate was 10. That is, ten molecules of DOCA werecoupled to one molecule of heparin. The dose amount was 200 mg/kg. At 1,2, and 3 hours after dosing, rats were anesthetized with diethyl etherand were sacrificed by cutting the diaphragm. Gastric, duodenal,jejunal, and ileal tissues were removed from the rats and fixed inneutral buffered formalin for processing. GI tissues sampled beforeadministration of heparin-DOCA conjugate were prepared as controlsamples. The tissue specimens were washed with alcohol to remove anywater. Specimens were perfused with colored silicone and embedded inparaffin. The embedded specimens were cut into 5 μm sections using amicrotome at −20° C., and picked up on a glass slide. The tissuesections were then washed with xylene and absolute alcohol,respectively, to remove the paraffin. The prepared 5 μm sections werethen stained with hematoxylin and eosin (H&E) according to procedureswell known in the art. At least 4 rats were used for each treatment.

[0066] For evaluation by transmission electron microscopy (TEM), thegastric, duodenal, jejunal, and ileal tissues were fixed with 1% osmiumtetroxide in PBS (0.1 M, pH 7.4), and then hydrated by changing thealcohol concentration gradually from 50 to 100%. The hydrated tissueswere infiltrated with propylene oxide and embedded with an epon mixture.The embedded tissues were sectioned as about 50-60 nm thickness slides.These slides were stained very lightly with uranyl acetate and leadcitrate for 1 minute, and were observed by TEM (Hitachi 7100, Tokyo,Japan).

[0067]FIG. 5 shows that there was no evidence of damage to the GI wall,such as occasional epithelial cell shedding, villi fusion, congestion ofmucosal capillary with blood, or focal trauma, in any parts of thestomach, duodenum, jejunum, or ileum. These results confirm thatincreased absorption of heparin derivatives was not caused by thedisruption of the gastrointestinal epithelium.

[0068]FIG. 6 shows the electron-microscopic morphology of microvilliafter exposure to heparin derivatives. The control samples showedhealthy tight junctions, microvilli, and mitochondria. After 1, 2, and 3hours, the cell appearance in all sections showed on signs of damage,such as microvilli fusion, dissolution, disoriented cell layer withporosity, or cytotoxic effect. Microvilli exposed to heparin derivativeswere also found to be as healthy as the control. The absence of tissuedamage indicates that the enhancing effect of the coupled DOCA onheparin absorption in the GI tract was not caused by changing the tissuestructure.

EXAMPLE 14

[0069] Conjugation of Lower Molecular Weight Heparin to DOCA. Conjugatesof heparin to DOCA were synthesized according to the procedure ofExample 1 except that unfractionated heparin (“UFH”), i.e., the compoundreferred to simply as “heparin” in previous examples, 6000 molecularweight heparin (“LMWH(6K)”), and 3000 molecular weight heparin(“LMWH(3K)”) were used. The resulting conjugates, UFH-DOCA,LMWH(6K)-DOCA, and LMWH(3K)-DOCA, were then characterized, as shown inTable 2. TABLE 2 Absolute Absolute Mole Activity Activity Ratio ofMolecular (IU/mg) (IU/mg) DOCA to Conjugate Weight^(a) by aPTT by FXaHeparin LMWH(3K) 2,910  50.8 ± 4.9 124.7 ± 0.8 N/A LMWH(3K)- 3,410  40.0± 7.7 121.5 ± 1.6 1.3 DOCA LMWH(6K) 6,150 127.1 ± 2.4 148.4 ± 0.2 N/ALMWH(6K)- 7,576 108.5 ± 4.9 134.3 ± 0.8 3.6 DOCA UFH 12,386 184 167 N/AUFH-DOCA 16,320 128.8 ± 2.3 116.9 ± 0.5 10

[0070] The maximum ratio of DOCA to heparin obtained in UFH-DOCA was 10when the feed ratio of UFH to DOCA was 1:200. Under these conditions,the ratios obtained with lower molecular weight heparins were 1.3 forLMWH(3K)-DOCA and 3.6 for LMWH(6K)-DOCA. The mole ratio of DOCA toheparin decreased with the decrease in molecular weight of heparinbecause of the fewer number of amine groups available for bonding toDOCA. Bioactivities of the heparin-DOCA conjugates also decreased withthe decrease of molecular weight of heparin, although all heparin-DOCAconjugates demonstrated similar bioactivities in the range of 116.9+1.6to 134.3+0.8 by FXa assay. After conjugation with DOCA, all of theheparin-DOCA conjugates showed above 70% relative bioactivity comparedto the unmodified heparin.

EXAMPLE 15

[0071] Oral Absorption of Lower Molecular Weight Heparin-DOCAConjugates. The lower molecular weight heparin-DOCA conjugates preparedin Example 14 were tested for absorption in the GI tract of rats afteroral administration according to the procedure of Example 10. FIG. 7shows the effect of molecular weight of heparin on the absorption ofheparin-DOCA conjugates in the GI tract. LMWH(3K)-DOCA, LMWH(6K)-DOCA,and UFH-DOCA (i.e., heparin-DOCA) were each administered by oral gavageat 100 mg/kg dosage. The clotting times of LMWH(3K)-DOCA and UFH-DOCAwere lower than that of LMWH(6K)-DOCA; the mean aPTT times at 1 hourwere 31.0+6.01 and 51.0±8.7, respectively (p<0.005). These data suggestthat the clotting time of LMWH(6K)-DOCA was 1.5- and 3-fold greater thanthose of LMWH(3K)-DOCA and UFH-DOCA, respectively. The concentrationprofiles of heparin-DOCA conjugates with time were similar to theresults of the APTT assay. When UFH-DOCA was administered at 100 mg/kgdosage, the peak concentrations of plasma was 4.10+1.3 μg/ml, which wasvery low compared to the concentration of LMWH(6K)-DOCA at the samedosage level.

[0072] The absorption of LMWH(6K)-DOCA in the GI tract was determinedaccording to the dose amount in the range of 20 to 100 mg/kg, as shownin FIG. 8. When 100 mg/kg of LMWH(6K) was administered orally to rats,the clotting time as measured by aPTT assay was about 30 seconds at 1hour after dosing. This curve fell to baseline at 2 hours after dosing.On the other hand, oral delivery of LMWH(6K)-DOCA resulted in theincreased heparin absorption in rats as shown by the highly elevatedaPTT values. When LMWH(6K)-DOCA was dosed at 100 mg/kg, the peak plasmaaPTT value was about 87.8+11.1 seconds (the baseline aPTT valuesaveraged 20 seconds). Heparin derivatives dosed at 20 mg/kg and 50 mg/kggave mean peak aPTT responses of 52.5±4.7 and 68.4+7.2 seconds,respectively (p<0.005). The therapeutic range of heparin, which is about1.5-2.5 times baseline in aPTT, is matched with a dose of 20 mg/kg, asshown in FIG. 8A.

[0073] Concentrations of heparin derivatives in the plasma could bedetermined using the anti-FXa assay. When 100 mg/kg of LMWH(6K)-DOCA wasadministered orally, the concentration of LMWH(6K) was 1.34+0.28 μg/ml.The low concentration of LMWH(6K) in the plasma could not facilitateanticoagulation activity. However, the maximum peak of LMWH(6K)-DOCA was8.21+1.6 μg/ml at a dose of 100 mg/kg, as shown in FIG. 8B. Thetherapeutic target range was 0.1 to 0.2 IU/ml. The mean concentrationpeaks were about 9-10 times the baseline. These results suggest thatheparin derivatives can perform as an oral anticoagulant drug forpatients at risk for deep vein thrombosis and pulmonary embolism.

EXAMPLE 16

[0074] Histological Evaluation of the GI Tract after Oral Administrationof Lower Molecular Weight Heparin-DOCA Conjugates. GI tract tissues fromrats given a single dose of 100 mg/kg of lower molecular weightheparin-DOCA conjugates prepared according to the procedure of Example14 were examined histologically according to the procedures of Example13. The results were substantially similar to those of Example 13. Thatis, no evidence of damage to any of the tissues of the GI wall weredetected.

The subject matter claimed is:
 1. A method of treating a patient in needof anticoagulation therapy comprising orally administering an effectiveamount of a composition comprising heparin covalently bonded to ahydrophobic agent selected from the group consisting of bile acids,sterols, and alkanoic acids, and mixtures thereof.
 2. The method ofclaim 1 wherein said hydrophobic agent is a bile acid selected from thegroup consisting of cholic acid, deoxycholic acid, chenodeoxycholicacid, lithocholic acid, ursocholic acid, ursodeoxycholic acid,isoursodeoxycholic acid, lagodeoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid,dehydrocholic acid, hyocholic acid, hyodeoxycholic acid, and mixturesthereof.
 3. The method of claim 2 wherein said bile acid is deoxycholicacid.
 4. The method of claim 1 wherein said hydrophobic agent is asterol selected from the group consisting of cholestanol, coprostanol,cholesterol, epicholesterol, ergosterol, ergocalciferol, and mixturesthereof.
 5. The method of claim 1 wherein said hydrophobic agent is analkanoic acid comprising about 4 to 20 carbon atoms.
 6. The method ofclaim 5 wherein said alkanoic acid is a member selected from the groupconsisting of butyric acid, valeric acid, caproic acid, caprylic acid,capric acid, lauric acid, myristic acid, palmitic acid, stearic acid,and mixtures thereof.
 7. The method of claim 1 wherein said compositionfurther comprises a pharmaceutically acceptable carrier.
 8. The methodof claim 1 wherein said heparin comprises a molecular weight of at leastabout
 3000. 9. The method of claim 8 wherein said heparin comprises amolecular weight of at least about
 6000. 10. The method of claim 1wherein said heparin comprises a molecular weight less than about12,000.
 11. A method for enhancing oral administration of amacromolecular agent comprising: (a) conjugating said macromolecularagent to a hydrophobic agent selected from the group consisting of bileacids, sterols, alkanoic acids, and mixtures thereof to result in ahydrophobized macromolecular agent; and (b) orally administering aneffective amount of said hydrophobized macromolecular agent to a patientin need thereof.
 12. The method of claim 11 wherein said hydrophobicagent is a bile acid selected from the group consisting of cholic acid,deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursocholicacid, ursodeoxycholic acid, isoursodeoxycholic acid, lagodeoxycholicacid, glycocholic acid, taurocholic acid, glycodeoxycholic acid,glycochenodeoxycholic acid, dehydrocholic acid, hyocholic acid,hyodeoxycholic acid, and mixtures thereof.
 13. The method of claim 12wherein said bile acid is deoxycholic acid.
 14. The method of claim 11wherein said hydrophobic agent is a sterol selected from the groupconsisting of cholestanol, coprostanol, cholesterol, epicholesterol,ergosterol, ergocalciferol, and mixtures thereof.
 15. The method ofclaim 11 wherein said hydrophobic agent is an alkanoic acid comprisingabout 4 to 20 carbon atoms.
 16. The method of claim 15 wherein saidalkanoic acid is a member selected from the group consisting of butyricacid, valeric acid, caproic acid, caprylic acid, capric acid, lauricacid, myristic acid, palmitic acid, stearic acid, and mixtures thereof.17. The method of claim 11 wherein said macromolecular agent is a memberselected from the group consisting of heparin, heparan sulfate, sulfonylpolysaccharide, polysaccharide derivatives, and mixtures thereof. 18.The method of claim 17 wherein said macromolecular agent is heparin. 19.The method of claim 11 wherein said macromolecular agent is a peptide.20. The method of claim 19 wherein said macromolecular agent is insulin.21. The method of claim 19 wherein said macromolecular agent iscalcitonin.
 22. A method of treating a patient in need ofanticoagulation therapy comprising orally administering an effectiveamount of a composition comprising a member selected from the groupconsisting of heparin, heparan sulfate, sulfonyl polysaccharide,heparinoids, and mixtures thereof covalently bonded to a hydrophobicagent selected from the group consisting of bile acids, sterols, andalkanoic acids, and mixtures thereof.